The essential nature of the ubiquitous 26-kilobase circular replicon of Borrelia burgdorferi

Abstract

The genome of the type strain (B31) of Borrelia burgdorferi, the causative agent of Lyme disease, is composed of 12 linear and 9 circular plasmids and a linear chromosome. Plasmid content can vary among strains, but one 26-kb circular plasmid (cp26) is always present. The ubiquitous nature of cp26 suggests that it provides functions required for bacterial viability. We tested this hypothesis by attempting to selectively displace cp26 with an incompatible but replication-proficient vector, pBSV26. While pBSV26 transformants contained this incompatible vector, the vector coexisted with cp26, which is consistent with the hypothesis that cp26 carries essential genes. Several cp26 genes with ascribed or predicted functions may be essential. These include the BBB29 gene, which has sequence homology to a gene encoding a glucose-specific phosphotransferase system component, and the resT gene, which encodes a telomere resolvase involved in resolution of the replicated telomeres of the linear chromosome and plasmids. The BBB29 gene was successfully inactivated by allelic exchange, but attempted inactivation of resT resulted in merodiploid transformants, suggesting that resT is required for B. burgdorferi growth. To determine if resT is the only cp26 gene essential for growth, we introduced resT into B. burgdorferi on pBSV26. This did not result in displacement of cp26, suggesting that additional cp26 genes encode vital functions. We concluded that B. burgdorferi plasmid cp26 encodes functions critical for survival and thus shares some features with the chromosome.

FIG. 1.

Graphic representation of the B. burgdorferi strain B31 cp26 plasmid. The approximate sizes and orientations of the 29 ORFs carried by cp26 are indicated. Genes on cp26 that exhibit homology to genes whose functions are known or that are of significant interest are labeled and indicated by arrows having different colors. Genes on cp26 whose functions are unknown are indicated by gray arrows. PTS, phosphotransferase system.

FIG. 2.

(A) Shuttle vector derived from cp26 (pBSV26). (B) cp26-derived shuttle vector carrying the resT gene (pBSV26resT). Relevant restriction sites are indicated. ColE1, E. coli origin and replication; ZEO, zeocin resistance marker; flgB_p::kan, kanamycin resistance marker fused to the flgB promoter (6).

FIG. 3.

Southern blot analysis of B. burgdorferi pBSV26 transformants. Wild-type B. burgdorferi DNA (lane 1), pBSV26 plasmid DNA isolated from E. coli (lane 2), and pBSV26 B. burgdorferi transformant DNA (lanes 3 to 8) were used. (A) Southern blot of genomic DNA was first probed with the kanamycin resistance gene. (B) Southern blot of genomic DNA was stripped and then probed with the cp26 gene ospC. An asterisk indicates the position of the endogenous, supercoiled form of cp26, while a solid square indicates the position of the supercoiled, extrachromosomal shuttle vector. A solid circle indicates the position of the supercoiled cp26 with the pBSV26 integrant, and a solid triangle indicates the position of the supercoiled cp26 dimer with the pBSV26 integrant. Unmarked higher-molecular-weight bands are linear forms of the circular plasmid that were a result of plasmid DNA preparation. The positions of DNA size standards (in kilobases) are indicated on the left.

FIG. 4.

Targeted inactivation of BBB29 and resT. (A) Organization of BBB29 and flanking genes on cp26. Also shown are the deletion of 469 bp of BBB29 and insertion of the kanamycin resistance cassette. The small arrows indicate the cp26 fragment used in the allelic exchange construct for inactivation of BBB29. (B) Organization of resT and flanking genes on cp26. Also shown are the deletion of 375 bp of resT and the insertion of the gentamicin resistance cassette (flgB_P::aacC1). Relevant restriction sites are indicated. The small arrows indicate the cp26 fragment used in the allelic exchange construct for inactivation of resT.

FIG. 5.

Southern blot analysis of transformants with a resT inactivation construct. Wild-type B. burgdorferi (wt) and XL-resTΔ transformant (tx) plasmid DNA were digested with BglI, which cut once within cp26 and linearized the plasmid, and with NcoI and SpeI, whose restriction sites flanked the resT gene. (A) The blot was first probed with the gene that confers gentamicin resistance, aacC1. (B) The same blot was stripped and then probed with the resT gene. The positions of NcoI and SpeI fragments corresponding to the mutant and wild-type copies of resT are indicated. The positions of DNA size standards (in kilobases) are indicated on the left.

FIG. 6.

Southern blot analysis of pBSV26resT transformants. Wild-type B. burgdorferi DNA (lane 1), pBSV26resT plasmid DNA isolated from E. coli (lane 2), and pBSV26resT transformant DNA (lanes 3 to 8) were used. (A) Southern blot of genomic DNA was first probed with the kanamycin resistance gene. (B) The same Southern blot was stripped and then probed with the cp26 gene ospC. An asterisk indicates the position of the endogenous, supercoiled form of cp26, while a solid square indicates the position of the supercoiled, extrachromosomal shuttle vector pBSV26resT. A solid circle indicates the position of the supercoiled cp26 monomer with a pBSV26resT integrant, and a solid triangle indicates the position of a cp26 dimer with a pBSV26resT integrant. Unmarked higher-molecular-weight bands are linear forms of the circular plasmid that were a result of plasmid DNA preparation. The positions of DNA size standards (in kilobases) are indicated on the left.